Isolation and characterization of Flexilinea flocculi gen. nov., sp. nov., a filamentous, anaerobic bacterium belonging to the class Anaerolineae in the phylum Chloroflexi.

A novel obligately anaerobic bacterium, designated strain TC1T, was isolated from methanogenic granular sludge in a full-scale mesophilic upflow anaerobic sludge blanket reactor treating high-strength starch-based wastewater. Cells had a multicellular filamentous morphology, stained Gram-negative and were non-motile. The filaments were flexible, generally >100 μm long and 0.3-0.4 μm wide. Growth of the isolate was observed at 25-43 °C (optimum 37 °C) and pH 6.0-8.5 (optimum pH 7.0). Strain TC1T grew chemo-organotrophically on a range of carbohydrates under anaerobic conditions. Yeast extract was required for growth. The major fermentative end products of glucose, supplemented with yeast extract, were acetate, lactate, succinate, propionate, formate and hydrogen. Co-cultivation with the hydrogenotrophic methanogen Methanospirillum hungatei DSM 864T enhanced growth of the isolate. The DNA G+C content was determined experimentally to be 42.1 mol%. The major cellular fatty acids were anteiso-C15 : 0, iso-C15 : 0 and iso-C17 : 0 3-OH. Based on 16S rRNA gene sequence analysis, strain TC1T belonged to the class Anaerolineae in the phylum Chloroflexi, in which Ornatilinea apprima P3M-1T was its closest phylogenetic relative (88.3 % nucleotide identity). Phylogenomic analyses using 38 and 83 single-copy marker genes also supported the novelty of strain TC1T at least at the genus level. Based on phylogenetic, genomic and phenotypic characteristics, we propose that strain TC1T represents a novel species of a new genus, for which we suggest the name Flexilinea flocculi gen. nov., sp. nov. The type strain of Flexilinea flocculi is strain TC1T ( = JCM 30897T = CGMCC 1.5202T).